Muscarinic cholinergic receptors (mAChRs) are present in high concentrations in the central nervous system and have been implicated in the etiology underlying a number of neuropsychiatric disorders. At least four mRNAs encoding mAChR subtypes are now known to exist and each gene product has its own distinct structural and pharmacological characteristics. While two of these subtypes (M1 and M2) have received considerable attention, relatively little is known of the properties of the other two and their mode of interaction with effector mechanisms. The human neuroblastoma cell line SK-N-SH expresses a large number of high MW mAChRs of the M3 subtype as defined pharmacologically. These receptors couple to enhanced phosphoinositide breakdown and Ca2+ signaling, but not to the inhibition of adenylate cyclase. The objectives of this proposal are three-fold. 1) To determine the contribution of glycosylation to the structural and functional characteristics of the receptor. Lectin affinity chromatography and endoglycosidase treatments will determine the extent of glycosylation of the receptor, while results obtained with cells grown in the presence of inhibitors of glycosylation will point to its functional significance. 2) To determine which guanine- nucleotide (G)-binding proteins can functionally interact with the M3 receptor and to probe the identity of the protein (Go), specifically linked to activation of phospholipase C. Using reconstitution assays, we will determine which G-proteins can restore high affinity agonist binding when added to membranes previously depleted of G-proteins. The identity of Gp will be assessed from the ability of muscarinic agonists to increase the labeling of un from guanine nucleotides. 3) To determine the relationship between mass of inositol trisphosphate (IP3) in cells and the IP3 requirements for occupancy of IP3 receptors and mobilization of Ca2+. A role for IP3 in mediation of Ca2+ influx will be assessed by measurement of IP3 binding sites in-plasma membranes and an analysis of their respective time-courses, agonist specificities and the effects of antagonists. The availability of a homogenous population of human neuronal cells that express an abundance of the M3 subtype provides a unique opportunity of studying this specific receptor subtype. It is likely that these studies will form the basis of an understanding of their physiological role and possible relevance to CNS disorders.